Bivariate flow cytogenetics was used to determine the DNA content of the Y chromosome in male C57BL/6J mice. The DNA content of the Y chromosome was estimated by mixing chromosomes from male C57/BL/6J mice with those from the human cell line GM130 prior to flow cytogenetic analysis. It was found that the C57BL/6J Y chromosome is similar in DNA content to human chromosome 16. On the basis of these results, we estimate that the C57BL/6J chromosome must contain at least 92 Mb of DNA, considerably more than previously reported.